In recent years, the number of diabetic patients has been increasing as the diet has become richer. To prevent complications in diabetic patients, blood sugar levels need to be maintained at the levels close to those of healthy individuals, and apparatus for self-measurement of blood sugar levels are widely used so that patients can monitor the blood sugar levels themselves at home. However, since blood sugar levels vary depending on the meal, and the measurement has to be done frequently, patients suffer from a heavy burden. It is also difficult for patients to correctly interpret measured values due to lack of knowledge, and it is not easy to strictly control blood sugar levels.
Meanwhile, since 1,5-anhydroglucitol is not affected by meal and reflect the blood sugar control in diabetic patients over the past one week, diabetic patients can correctly understand their blood sugar control by “once weekly” measurement at home alone. A 1,5-anhydroglucitol self-measurement kit would provide a great advantage for patients, but conventional methods for measuring 1,5-anhydroglucitol using serum or plasma as a specimen require blood cell separation and are not suitable for self-measurement because the measurement requires a large amount of a specimen. Therefore, 1,5-anhydroglucitol self-measurement kits, including those using a trace amount of whole blood as it is as a specimen, have not been realized.
The 1,5-anhydroglucitol measuring methods described in PATENT DOCUMENTS 1 to 9 use serum or plasma as a specimen, not whole blood, and do not involve electrochemical measurement.
Furthermore, when a whole blood specimen is used for the 1,5 AG Kit for Animals (Nippon Kayaku Co., Ltd.) distributed by Wako Pure Chemical Industries, Ltd., blood is hemolysed by adding purified water or an aqueous solution of 10 mM ethylenediamine tetraacetic acid (EDTA) and further centrifuged, the supernatant is treated with a column, and 1,5-anhydroglucitol is measured by a colorimetric method using a pigment. In other words, this method is not a method for electrochemically measuring 1,5-anhydroglucitol using whole blood as it is as a specimen without separating blood cells.
The reason for conventionally using serum or plasma as a specimen, not whole blood, is that glucose is also contained in blood cells. Even when a reagent for eliminating or converting glucose is added to whole blood, glucose remains in blood cells without being eliminated or converted. Since glucose is released through the cell membrane in the step of measuring 1,5-anhydroglucitol in blood, interference by glucose cannot be completely eliminated. In a reaction for detecting 1,5-anhydroglucitol in blood, interference by hemoglobin in erythrocytes having an oxidation-reduction ability is expected. So far, a method for measuring blood 1,5-anhydroglucitol using whole blood by enzymatically eliminating or converting glucose beforehand without separating blood cells is not known.
Furthermore, methods for electrochemically measuring 1,5-anhydroglucitol are described in NON-PATENT DOCUMENT 1 and PATENT DOCUMENTS 10 and 11. However, NON-PATENT DOCUMENT 1 describes a method for measuring 1,5-anhydroglucitol in urine using an enzyme sensor composed of a hydrogen peroxide electrode and an enzyme-fixed membrane. However, measurement of 1,5-anhydroglucitol in whole blood, in which glucose and the like coexist, is not mentioned. In PATENT DOCUMENT 10, 1,5-anhydroglucitol is measured by amperometry using a dehydrogenase and phenazine methosulfate as an electron acceptor. However, only examples for the reference standard of 1,5-anhydroglucitol are mentioned, and there is no description about the measurement in whole blood. In PATENT DOCUMENT 11, an enzyme having an ability of oxidizing 1,5-anhydroglucitol is allowed to act, and the produced hydrogen peroxide is electrochemically measured using a hydrogen peroxide electrode. However, serum is used in this measurement as well, and this is not a measurement method using whole blood.
Meanwhile, 1,5-anhydroglucitol is a compound which is a reduced glucose at the position of 1 and has a chemical structure very similar to that of glucose. Therefore, many of enzymes used for measurement of 1,5-anhydroglucitol also react with glucose. Blood contains glucose 20 times or more abundant than 1,5-anhydroglucitol. Therefore, to measure 1,5-anhydroglucitol, glucose must be eliminated or converted in some way so that glucose should not react with enzymes for measuring 1,5-anhydroglucitol. Furthermore, when glucose derivatives produced by this conversion react with enzymes for measuring 1,5-anhydroglucitol, these derivatives must also be eliminated or converted.
Glucose is eliminated or converted by oxidizing glucose with glucose oxidase or phosphorylating glucose with hexokinase in PATENT DOCUMENT 1, oxidizing glucose with glucose oxidase and gluconolactonase or glucose dehydrogenase and gluconolactonase in PATENT DOCUMENT 2, converting glucose to fructose-1,6-diphosphate with hexokinase, phosphohexose isomerase, and 6-phosphofructokinase or glucose isomerase, fructokinase, and 6-phosphofructokinase in PATENT DOCUMENTS 3 and 4, phosphorylating glucose with glucokinase or hexokinase in PATENT DOCUMENT 5, and phosphorylating glucose with an enzyme that phosphorylates glucose to glucose-6-phosphate in PATENT DOCUMENT 6, and then 1,5-anhydroglucitol is measured. In these documents, glucose is converted to glucono-1,5-lactone, glucose-6-phosphate, gluconic acid, fructose-6-phosphate, fructose-1,6-diphosphate, or the like. So far, however, measurement by eliminating or converting glucose in whole blood with an enzyme and then quantifying electrochemically 1,5-anhydroglucitol in whole blood with an enzyme is not known.    PATENT DOCUMENT 1: Japanese Patent No. 2983015    PATENT DOCUMENT 2: JP-A-2001-78797    PATENT DOCUMENT 3: Japanese Patent No. 3170320    PATENT DOCUMENT 4: Japanese Patent No. 3217180    PATENT DOCUMENT 5: JP-A-2001-116756    PATENT DOCUMENT 6: Japanese Patent No. 2872983    PATENT DOCUMENT 7: JP-A-63-185397    PATENT DOCUMENT 8: JP-A-10-191998    PATENT DOCUMENT 9: JP-A-8-70893    PATENT DOCUMENT 10: JP-A-7-67697    PATENT DOCUMENT 11: JP-A-62-79780    PATENT DOCUMENT 12: JP-A-5-304997    PATENT DOCUMENT 13: JP-A-63-22185    PATENT DOCUMENT 14: JP-A-2-268679    PATENT DOCUMENT 15: JP-A-2000-135079    PATENT DOCUMENT 16: JP-A-11-18760    PATENT DOCUMENT 17: JP-A-2000-175698    PATENT DOCUMENT 18: JP-A-10-179140    NON-PATENT DOCUMENT 1: Biomed. Chromatogr., vol. 7, p. 41 (1993)